Production of High Levels of DHA in Microalgae Using Modified Amounts of Chloride and Potassium

ABSTRACT

Methods for production of highly unsaturated fatty acids by marine microorganisms, including the heterotrophic marine dinoflagellate  Crypthecodinium , using low levels of chloride ion are disclosed. Specifically, methods of increasing production of highly unsaturated fatty acids by marine microorganisms while growing in low chloride media by manipulating sodium ion and potassium ion levels. The invention also relates to methods of production of highly unsaturated fatty acids by marine organisms at low pH levels, and includes methods for generation of low pH tolerant strains.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/313,524, filed Dec. 20, 2005, which is a divisional of U.S. patentapplication Ser. No. 10/957,075, filed Oct. 1, 2004, now U.S. Pat. No.7,163,811, which claims the benefit of priority under 35 U.S.C. §119(e)from U.S. Provisional Application Ser. No. 60/508,505, filed Oct. 2,2003. Each of the above-identified patent applications is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

This invention generally relates to methods for production of highlyunsaturated fatty acids by marine microorganisms using modified amountsof chloride and potassium ion in the culture medium. More specifically,the invention is directed to a process for producing high levels ofdocosahexaenoic acid (DHA) by culturing marine microalgae, including theheterotrophic marine dinoflagellate, Crypthecodinium, in fermentorsunder non-corrosive conditions which includes culturing in a lowchloride ion and a high potassium ion environment. This invention alsorelates to methods for production of highly unsaturated fatty acids,including DHA, by marine microorganisms at low pH levels.

BACKGROUND OF THE INVENTION

The beneficial effects of increased dietary intake of long chain omega-3fatty acids in humans has been well documented, which includes thereduction of cardiovascular and inflammatory diseases (i.e. arthritisand atherosclerosis), reduction of depression, increasing length ofgestation in the third trimester, and inhibiting tumor growth. Severalheterotrophic marine microorganisms have been found to produce highlevels of these important essential fatty acids, including that of genusCrypthecodinium (Jiang and Chen, Process Biochemistry 35 (2000)1205-1209; Jiang and Chen, Journal of Industrial Microbiology &Biotechnology, (1999) Vol. 23, 508-513; Vazhappilly and Chen, Journal ofthe American Oil Chemists Society, (1998) Vol. 75, No. 3 p 393-397;Kyle, U.S. Pat. No. 5,407,957; U.S. Pat. No. 5,397,591; U.S. Pat. No.5,492,938; and U.S. Pat. No. 5,711,983).

Crypthecodinium cohnii is one of the most desirable organisms to utilizefor the production of DHA (C22:6n-3), one of the most important longchain omega-3 fatty acids. C. cohnii is advantageous because DHA is theonly polyunsaturated fatty acid (PUFA) produced by this organism inappreciable quantities. Other organisms produce two or morepolyunsaturated fatty acids (PUFAs) in their lipids, and the complexityof their lipid profile can limit the use of their oils in some food andpharmaceutical applications (e.g. due to the presence of otherundesirable PUFAs in the oil or due to ratios of the different PUFAsfalling out of the desirable range for the specific application). In themarine environment, Crypthecodinium cohnii is usually found in fullsalinity seawater and, as such, is adapted to growth in an environmentwith a high chloride concentration. In fact, most cultures in publishedresearch on C. cohnii show that the growth and DHA production does bestat salinities greater than about 20% of seawater (Jiang and Chen). Thechloride ion concentration equivalent to 20% seawater is about 3870 ppmchloride ion or 3.87 g/l chloride ion. (Home 1969).

Tuttle and Loeblich (1975) developed an optimal growth medium for C.cohnii. The disclosed medium contained a sodium chloride concentrationof 342 millimolar (mM). The equivalent grams per liter of sodium ion andchloride ion in a 342 mM sodium chloride solution are 7.86 g/L sodiumion and 12.12 g/L of chloride ion.

Beach & Holz (1973) reported that when culturing C. cohnii over a rangeof NaCl concentrations (0.3%, 1.8% and 5.0% (1.82 g/l, 10.9 g/l and 30.3g/l chloride ion, respectively)) lipid yield (expressed as mg per 10⁹cells) declined as NaCl concentrations decreased. Lipid yield at 0.3%NaCl was approximately one third of that at 5.0% NaCl. More recently,Jiang and Chen (1999) determined the effects of salinity on cell growthand DHA content with three strains of Crypthecodinium cohnii and foundin all cases that the optimum growth rates for cells and DHA yields werebetween 5 g/L and 9 g/L sodium chloride, which corresponds to 3.0 and5.5 g/L chloride ion, respectively.

The natural chloride concentration of seawater (19,353 ppm, or 19.35 g/lchloride ion) (Home 1969, page 151) promotes corrosion in stainlesssteel fermentors. For example, of the two common grades of stainlesssteel used in manufacturing fermentors, 304-stainless steel issusceptible to corrosion when the chloride level exceeds 300 ppm (0.3g/l chloride ion), and 316-stainless steel is susceptible to corrosionwhen the chloride level exceeds 1000 ppm (1 g/l chloride ion). Othergrades of stainless steel exist that are more resistant to chloridecorrosion, but they are extremely expensive and generally only used infermentation equipment employed for the production of very expensivecompounds.

Although it may be predicted that minimizing corrosion of stainlesssteel fermentors may be achieved by lowering chloride concentrations inthe culture medium, in practice this is not an easy task. Marinemicroalgae, which are derived from the sea, generally require a certainamount of chloride ion, preferably as sodium chloride, to maintaingrowth and lipid production when grown in culture.

However, attempts to date to grow marine microalgae at low chlorideconcentrations while maintaining levels of production of omega-3polyunsaturated fatty acids such as DHA have been unsuccessful. Jiangand Chen (1999) were unable to demonstrate significant DHA yields atNaCl levels less than 5 g/L, corresponding to a chloride level of about3033 ppm or 3 g/L.

U.S. Pat. No. 6,410,281, issued Jun. 25, 2002, to Barclay, provides amethod for growing euryhaline organisms such as Thraustochytrium sp. andSchizochytrium sp. in low chloride media by substituting non-chloridesodium salts to replace the sodium lost when lowering sodium chloridelevels.

There exists a need for a process which would enable the production of ahigh yield of DHA from Crypthecodinium cohnii, while inhibiting orpreventing corrosion in the most commercially desirable productionvessels, stainless steel culture fermentors. This process would have toenable effective growth of the microorganism in a medium containingpreferably less than 300 ppm chloride. Three hundred ppm chloriderepresents a level 10-18 times lower than the lowest chloride levelsdemonstrated by Jiang & Chen (1999) to be the best for the production ofstrains of Crypthecodinium.

Another desirable characteristic of microbial fermentations is theability to grow cells at low pH (less than or equal to about pH=5.0) toinhibit the growth of bacteria in fungal fermentations. However, theliterature indicates that Crypthecodinium grows best at a neutral pH(about pH 7). Tuttle and Loeblich in Phycologia Vol. 14(1) 1-8 (1975),disclose that the pH optimum for Crypthecodinium growth is 6.6, withgrowth being “very slow” below pH 5.5. There exists a need for strainsand/or methods of growing Crypthecodinium at low pH while retainingnormal growth and production of DHA.

SUMMARY OF THE INVENTION

In attempting to minimize sodium chloride levels in culture medium forCrypthecodinium, where sodium chloride leads to the problem of corrosionof fermentors, the inventors have surprisingly discovered that sodiumchloride levels can be reduced by manipulation of the sodium andpreferably the potassium salts in the culture medium to compensate forthe decrease of chloride ion (down to 300 ppm or 0.3 g/L chloride ion)while maintaining the yield of DHA similar to what is obtained at about4.5 g/L NaCl (corresponding to 2.73 g/l chloride ion).

The present inventors have identified culture conditions that allowCrypthecodinium to be grown in medium with substantially loweredchloride levels (down to about 0.3 g/l chloride ion) without adverselyaffecting the city weight, fat content or DHA content when compared togrowth in a normal “high chloride” medium. Attaining a comparable DHAyield was not merely a matter of replacing the sodium chloride in themedium with other sodium salts. In fact, replacement of sodium chloridewith an equivalent amount of sodium from other sodium salts (i.e. sodiumsulfate) did not result in a DHA yield comparable to the high chloridecontrol case, but actually resulted in a further decrease in the DMAyield of the culture. Instead the present inventors surprisingly foundthat the best DMA yield was obtained when the potassium concentration(relative to that in seawater at 4.5 g/l NaCl sir 17% of seawater) wassignificantly increased. It is unexpected that a substantial decrease inthe amount of sodium and an increase in potassium concentration would beeffective in compensating for a reduction in the chloride content of themedium.

In one embodiment, the present invention includes a method for producingdocosahexaenoic acid (DHA) by culturing heterotrophic microalgae of theclass Dinophyceae in a culture medium. The medium comprises chloride ionat a concentration of less than or equal to about 2 g/l and potassiumion at a concentration of greater than or equal to about 0.25 g/l. Inthis embodiment, the microalgae produces at least about 0.04 g DHA perliter of 7 day culture. A 7 day culture generally has about 5×10⁶cells/ml or about 5×10⁹ cells/liter. Therefore, a culture having about0.2 g/l DHA at 7 days contains about 0.04 g DHA/10⁹ cells, In apreferred embodiment, the microalgae is of the genus Crypthecodinium. Amore preferred microalgae is Crypthecodinium cohnii. Preferably, thechloride ion concentration is less than or equal to about 1 g/l, evenmore preferably less than or equal to about 0.3 g/l. Preferably, thepotassium ion is greater than or equal to about 0.4 g/l, and even morepreferably is equal to or greater than about 0.8 g/l. Preferably, thesource of potassium ion is potassium sulfate. In a preferred embodiment,the medium further comprises a source of sodium ion such that the sodiumion concentration is from about 1 g/l to about 8 g/l. More preferably,the sodium ion is from about 1.5 g/l to about 5 g/l. A preferred sourceof sodium ion is sodium sulfate. Included in the present invention is abiomass produced by this method.

In another embodiment, the present invention includes a method ofproducing DHA by culturing heterotrophic microalgae of the classDinophyceae in a culture medium. The medium comprises chloride ion at aconcentration of less than or equal to about 2 g/l, potassium ion at aconcentration of greater than or equal to about 0.25 g/l and sodium ionpresent in a ratio of less than or equal to about 27:1 wt:wtsodium:potassium. In this embodiment, the microalgae produces at leastabout 0.2 g DHA per liter 7 day culture or 0.04 g DHA/10⁹ cells. In apreferred embodiment, the microalgae is of the genus Crypthecodinium. Amore preferred microalgae is Crypthecodinium cohnii. Preferably, thechloride ion concentration is less than or equal to about 1 g/l, evenmore preferably less than or equal to about 0.3 g/l. Preferably, thepotassium ion is greater than or equal to about 0.4 g/l, and even morepreferably is equal to or greater than about 0.8 g/l. Preferably, thesource of potassium ion is potassium sulfate. The medium furthercomprises a source of sodium ion such that the sodium ion is present inthe medium in a ratio of less than 27 times (by weight) the weight ofthe potassium ion (expressed as 27:1 sodium:potassium wt:wt.). In apreferred embodiment, the sodium:potassium ratio is less than about15:1. More preferred is a sodium:potassium ratio of about 4:1. Apreferred source of sodium ion is sodium sulfate. Included in thepresent invention is a biomass produced by this method.

The present inventors have also identified culture medium conditions andstrains that allow Crypthecodinium to be grown in medium withsubstantially lowered pH levels, while still maintaining a commerciallypractical rate of growth and production of lipid, including DHA. Inanother embodiment, the present invention includes a method of producingDHA by culturing heterotrophic microalgae of the class Dinophyceae in aculture medium, wherein the culture medium has a pH of less than about6, and wherein the microalgae produces at least about 0.04 g DHA/10⁹cells. The medium may further comprise chloride ion at a concentrationof less than or equal to about 2 g/l, potassium ion at a concentrationof greater than or equal to about 0.25 g/l and sodium ion present in aratio of less than or equal to about 27:1 wt:wt sodium:potassium. Inthis embodiment, the microalgae produces at least about 0.04 g DHA/10⁹cells. In a preferred embodiment, the microalgae is of the genusCrypthecodinium. A more preferred microalgae is Crypthecodinium cohnii.In a preferred embodiment, the pH is less than or equal to about pH 5.5,more preferably is less than or equal to about 5.0, and even morepreferably less than or equal to about 4.5. In a preferred embodiment,the medium further comprises chloride ion concentration at less than orequal to about 2 g/l, preferably less than or equal to about 1 g/l, evenmore preferably less than or equal to about 0.3 g/l. The medium alsocomprises potassium ion at concentrations greater than or equal to about0.25 g/l, greater than or equal to about 0.4 g/l, and even morepreferably is greater than or equal to about 0.8 g/l. Preferably, thesource of potassium ion is potassium sulfate. In a preferred embodiment,the medium further comprises a source of sodium ion such that the sodiumion concentration is from about 1 g/l to about 8 g/l. More preferably,the sodium ion is from about 1.5 g/l to about 5 g/l. A preferred sourceof sodium ion is sodium sulfate. Included in the present invention is abiomass produced by this method.

The present invention also includes a method for the selection of a lowpH tolerant heterotrophic microalgae of the class Dinophyceae,comprising subculturing said microalgae in low pH media until the yieldof DHA is greater than or equal to about 0.04 g DHA/10⁹ cells. In apreferred embodiment, the pH is less than or equal to about 6, is lessthan or equal to about 5, is less than or equal to about 4.5. Includedin the present invention is microalgae and a biomass produced by thismethod.

These and other objects, features, and advantages of the invention willbecome apparent from the following best mode description, the drawingsand the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation of a time course of DHA yield forduplicates of C. cohnii strain T-HF grown at pH 6.3 and 3 g/l chlorideion (denoted pH 6.3 SSM) and C. cohnii strain T-HF, adapted to low pH,grown at pH 5 and 1 g/l chloride ion (denoted pH 5.0 LCSSI).

FIG. 2 is a graphical representation of a time course of DHA yield forduplicates of C. cohnii strain T-HF grown at pH 6.3 and 3 g/l chlorideion (denoted pH 6.3 SSM) and C. cohnii strain T-HF, adapted to low pH,grown at pH 4.5 and 1 g/l chloride ion (denoted pH 5.0 LCSSI).

DESCRIPTION OF THE INVENTION

The present invention solves the above-identified problem of corrosionof fermentors caused by high sodium chloride levels used for the growthof marine microalgae of the class Dinophyceae. The inventors havediscovered culture media components which allow for commercially viablelevels of growth of marine microalgae of the class Dinophyceae andproduction of DHA under low sodium chloride conditions, by usingmodified amounts of chloride and potassium ion in the culture medium.More specifically, the inventors have discovered that the loss of sodiumcaused by reducing sodium chloride to non-corrosive levels may be atleast partially offset by increasing potassium levels in the culturemedia.

The present invention also solves the above-identified problem ofallowing for growth of a marine microalgae of class Dinophyceae whilesimultaneously discouraging growth of bacteria. More specifically, thepresent invention provides methods to culture marine organisms such thatthey become tolerant to low pH. The present invention also providesstrains of such microorganisms which are tolerant to low pH. LowpH-tolerant strains provided by the inventors can, at low pH levels,grow to cell densities and achieve DHA production levels comparable tothat achieved by strains growing at more neutral pH levels. This is onlyone example of the technology encompassed by the invention, as theconcepts of the invention can readily be applied to other productionorganisms and other desired PUFAs as described in detail below.

One embodiment of the present invention includes a method to producedocosahexanenoic acid (DHA) by culturing heterotrophic microalgae of theclass Dinophyceae in a culture medium which includes the followingcomponents: chloride ion at a concentration of less than about 2 g/L,and potassium ion at a concentration of greater than about 0.25 g/L,where the microalgae produce at least about 0.2 g of DHA per liter of7-day culture. The 7 day culture generally has 5×10⁶ cells/ml resultingin about 0.04 g DHA/10⁹ cells. In preferred embodiments, theheterotrophic microalgae produce at least about 0.04 g DHA/10⁹ cells, atleast about 0.06 g DHA/10⁹ cells, at least about 0.08 g DHA/10⁹ cells,at least about 0.10 g DHA/10⁹ cells, at least about 0.12 g DHA/10⁹cells, at least about 0.14 g DNA/10⁹ cells, at least about 0.16 gDHA/10⁹ cells, at least about 0.18 g DHA/10⁹ cells, at least about 0.20g DHA/10⁹ cells, at least about 0.22 g DHA/10⁹ cells, at least about0.24 g DHA/10⁹ cells, at least about 0.26 g DHA/10⁹ cells, at leastabout 0.28 g DHA/10⁹ cells, or at least about 0.30 g DHA/10⁹ cells. Asused herein, reference to a nutrient concentration in a culture mediumrefers to the concentration of nutrients in the medium at the beginningof the step of culturing, which includes any nutrients carried over fromprevious stages in the process, such as preparation of an inoculum.

Microorganisms suitable for the present invention include heterotrophicmicroalgae, which include members of the class Dinophyceae(dinoflagellates). A preferred member of this class is a member of thegenus Crypthecodinium. A preferred member of the genus Crypthecodiniumis C. cohnii. Crypthecodinium cohnii is an obligate heterotrophrequiring a reduced carbon source for growth, and contains a fatty acidprofile in which DHA is the only polyunsaturated fatty acid present inappreciable quantities. Suitable organisms can be obtained from a numberof publicly available sources, including by collection from the naturalenvironment. For example, the American Type Culture Collection currentlylists forty-five available strains of Crypthecodinium cohnii, identifiedas ATCC Nos. 30021, 30334-30348, 30541-30543, 30555-30557, 30571, 30572,30772-30775, 30812, 40750, 50050-50060, and 50297-50300. As used herein,any microorganism, or any specific type of organism, includes wildstrains, mutants, or recombinant types.

Apart from the sodium, chloride and potassium concentrations which arethe subject of the present invention and discussed more fully below,other components of media of the present invention can be any componentsknown in the art that promote the growth and production of DHA atcommercially practicable levels, and include components such as thosedisclosed in U.S. Pat. No. 5,130,242, U.S. Pat. No. 5,407,957, U.S. Pat.No. 5,397,591; U.S. Pat. No. 5,492,938; and U.S. Pat. No. 5,711,983, allof which are incorporated by reference herein in their entirety. Morespecifically, a source of carbon, such as glucose, various starches,molasses, ground corn and the like may be used. A source of assimilableorganic or inorganic nitrogen is also included in the culture media.Nitrogen sources may include nitrate, urea, ammonium salts, amino acidsand the like. A source of assimilable phosphorous may also be provided.The medium also may contain a source of microbial growth factors, whichare unspecified or specified compounds that enhance the heterotrophicgrowth of unicellular microorganisms, and may include yeast or otherextracts, soil extracts, and the like. Specific examples of growth mediafor C. cohnii and related organisms, for example, may also be found inJiang and Chen, Process Biochemistry 35 (2000) 1205-1209; Jiang andChen, Journal of Industrial Microbiology & Biotechnology, (1999) Vol.23, 508-513; Vazhappilly and Chen, Journal of the American Oil ChemistsSociety, (1998) Vol. 75, No. 3 p 393-397. Specific examples of preferredmedia to use with the present invention may be found in, for example,the Examples section herein below.

In one aspect of the media of the present invention, chloride ionconcentrations are present in a concentration of less than or equal toabout 2000 ppm or about 2 grams per liter of culture, more preferablyless than or equal to about 1.9 g/l, more preferably less than or equalto about 1.8 g/l, more preferably less than or equal to about 1.7 g/l,more preferably less than or equal to about 1.6 g/l, more preferablyless than or equal to about 1.5 g/l, more preferably less than or equalto about 1.4 g/l, more preferably less than or equal to about 1.3 g/l,more preferably less than or equal to about 1.2 g/l, more preferablyless than or equal to about 1.1 g/l, more preferably less than or equalto about 1.0 g/l, more preferably less than or equal to about 0.9 g/l,more preferably less than or equal to about 0.8 g/l, more preferablyless than or equal to about 0.7 g/l, more preferably less than or equalto about 0.6 g/l, more preferably less than or equal to about 0.5 morepreferably less than or equal to about 0.4 g/l, and most preferably lessthan or equal to about 0.3 g/l. In alternative embodiments, the minimumchloride concentration is at least about 0.025 g/l, at least about 0.05or at least about 0.1 g/l. The chloride ion component of the media ispreferably derived from a chloride salt, with a preferred salt beingsodium chloride. Other sources of chloride in the media includepotassium chloride and calcium chloride. Sources of chloride ion mayinclude more than one chloride-containing compound in the media, and mayinclude hydrochloric acid which may be used to adjust pH of the media,as well as MnCl₂ and FeCl₃.

In another aspect of the media of the present invention, the potassiumion concentration is greater than about 0.25 g/L. Potassium ion isgenerally present at low levels in seawater, being approximately 0.38g/l seawater. Culture media known in the art for growth of marinemicroalgae closely follows the composition of seawater, with levels ofpotassium ion generally being the same or less. For example, Tuttle andLoeblich (1975) disclose 9 mM KCl, which is the equivalent ofapproximately 0.35 g/l potassium ion. In Handbook of PhycologicalMethods (Janet R. Stein, Ed., Cambridge University Press, 1973),potassium ion in the media is disclosed to be 9.83 mM as potassiumchloride, which is the equivalent of approximately 0.36 g/l potassiumion. In one embodiment, the present invention includes potassium ion ata concentration of greater than about 0.39 g/l. The present inventorshave found that, once potassium ion is greater than a threshold level,cultures are relatively insensitive to the precise concentration ofpotassium ion, growing well and yielding commercially viable levels ofDHA at a range of potassium ion concentrations. Preferably, the lowerrange of potassium ion concentration is at least about 0.2 g/l, at leastabout 0.25 g/l, at least about 0.3 g/l, at least about 0.35 g/l, atleast about 0.4 g/l, at least about 0.45 g/l, at least about 0.5 g/l, atleast about 0.6 g/l, and at least about 0.7 g/l. Preferably, the upperrange of the potassium ion concentration is at most about 10 g/l, atmost about 6 g/l, at most about 4 g/l, at most about 3 g/l, at mostabout 2.8 g/l, at most about 2.6 g/l, at most about 2.4 g/l, at mostabout 2.2 g/l, at most about 2 at most about 1.9 g/l, at most about 1.8g/l, at most about 1.7 g/l, at most about 1.6 g/l, at most about 1.5g/l, and at most about 1 g/l. Most preferred concentrations of potassiumion are about 0.75 g/l, 0.8 g/l, 0.85 g/l, 0.9 g/l, and 0.95 g/l.Preferable ranges for potassium ion are between about 0.45 g/l and about1.5 g/l; more preferably between about 0.5 g/l and about 1.2 g/l; morepreferably between about 0.6 g/l and about 1 g/l; even more preferablybetween about 0.7 g/l and about 0.9 g/l; and most preferably about 0.8g/l.

The source of potassium ion can be from any potassium salt compatiblewith cell culture and microalgae of the class Dinophyceae in particular.Potassium ion may be derived from a mixture of salts in the media.Preferred potassium salts include potassium chloride, potassium sulfate,potassium acetate, potassium bicarbonate, potassium phosphate, amongothers. A preferred source of potassium ion is potassium sulfate.

In one aspect of the present invention, the amount of DHA yield from thecultures at harvest is greater than the amount of DHA yield fromcultures not grown in media of the present invention. In one embodiment,the DHA yield using low chloride concentrations using processes of thepresent invention is at least 0.2 gram DHA per liter of 7-day culture or0.04 g DHA/10⁹ cells.

In another aspect of the present invention, the media will also containadditional sources of sodium ion other than sodium chloride. The presentinventors have found that sodium ion levels are not critical to thepresent invention. Cultures of marine organisms of the present inventionare relatively insensitive to the precise concentration of sodium ion,growing well and yielding commercially viable levels of DHA at a rangeof sodium ion concentrations. Many different sources of sodium ion arecompatible with the present invention, including sodium sulfate, sodiumcarbonate, sodium hydrogen carbonate, and sodium acetate. A preferredsource of additional sodium ion is sodium sulfate. In a preferredembodiment, the medium contains at least about 1 g/l sodium ion up toabout 8 g/l sodium ion. At the lower end of the range, preferred sodiumion concentration is at least about 1 g/l, at least about 1.5 g/l, atleast about 2 g/l, and at least about 2.5 g/l. Preferably, the upperrange of the sodium ion concentration is at most about 15 g/l, at mostabout 12 g/l, at most about 10 g/l, at most about 9 g/l, at most about 8g/l, at most about 7 g/l, at most about 6 g/l, at most about 5.5 g/l, atmost about 5 g/l, at most about 4.5 g/l, at most about 4 g/l. Mostpreferred concentrations of sodium ion are about 2.75 g/l, 3 WI, 3.25g/l, 3.5 g/l, and 3.75 g/l. Preferable ranges for sodium ion are betweenabout 1.5 g/l up to about 7.5 g/l, even more preferred is about 2.0 g/lup to about 6 g/l, and even more preferred is more than about 2.5 g/l upto about 5 g/l. In the most preferred embodiments, sodium ion is atleast about 3 g/l to about 3.5 g/l. The most preferred level of sodiumis about 3.25 g/l. As described previously, the cultures are relativelyinsensitive to the precise levels of sodium, and therefore even higherlevels may be used. However, once sodium levels above about 8 g/l areused, the culture yields begin to drop slightly.

In another embodiment, the present invention includes a method ofproducing DHA by culturing heterotrophic microalgae of the classDinophyceae in a culture medium. The medium comprises chloride ion at aconcentration of less than or equal to about 2 g/l, potassium ion at aconcentration of greater than or equal to about 0.25 g/l and sodium ionpresent in a ratio of less than or equal to about 27:1 wt:wtsodium:potassium. In this embodiment, the microalgae produces at leastabout 0.2 g DHA per liter 7 day culture or 0.04 g DHA/10⁹ cells. In thisembodiment, the culture medium contains sodium ion in a ratio withpotassium ion of less than or equal to about 27:1, weight:weight. Inseawater, the sodium ion to potassium ion ratio is approximately 27.3:1.In other words, the amount of sodium ion is about 27.3 times higher thanthe amount of potassium ion. In the present invention, the inventorshave found that increasing the potassium ion relative to the sodium ionincreases the yield of DHA from the culture. A preferred ratio of sodiumion to potassium ion less than or equal to about 27:1, less than orequal to about 25:1, less than or equal to about 23:1, less than orequal to about 21:1, less than or equal to about 19:1. More preferredare ratios of less than or equal to about 17:1, less than or equal toabout 15:1, less than or equal to about 13:1, less than or equal toabout 11:1. Even more preferred are ratios of less than or equal toabout 9:1, less than or equal to about 7:1, or less than or equal toabout 5:1. A preferred ratio is about 4:1.

In another embodiment, the present invention includes a method ofproducing DHA by culturing heterotrophic microalgae of the classDinophyceae in a culture medium, wherein the culture medium has a pH ofless than about 6, and wherein the microalgae produces at least about0.2 g DHA per liter of 7 day culture or 0.04 g DHA/10⁹ cells. In apreferred embodiment, the pH is less than or equal to about 5.5, andmore preferably less than or equal to about 5. In a preferredembodiment, the pH is less than or equal to about 4.5. In a preferredembodiment, the medium further comprises chloride ion concentration atless than or equal to about 2 g/l, preferably less than or equal toabout 1 g/l, even more preferably less than or equal to about 0.3 g/l.The medium also preferably comprises potassium ion at concentrationsgreater than or equal to about 0.25 g/l, greater than or equal to about0.4 g/l, and even more preferably is greater than or equal to about 0.8g/l. Preferably, the source of potassium ion is potassium sulfate. In apreferred embodiment, the medium further comprises a source of sodiumion such that the sodium ion concentration is from about 1 g/l to about8 g/l. More preferably, the sodium ion is from about 1.5 g/l to about 5g/l. A preferred source of sodium ion is sodium sulfate. Included inthis embodiment is a biomass produced by this method.

In another embodiment, the present invention includes a method for thepreparation of low pH tolerant strains of species of the class ofDinophyceae and strains produced thereby. Methods include preparation oflow pH media and subculturing the desired Dinophyceae species until theculture produces a desired amount of DHA. Subculturing may be carriedout in the following manner. An inoculum of the desired Dinophyceaespecies is placed in the low pH media and allowed to grow a definedamount of time, preferably 7 days. The amount of time is not critical,but should be chosen such that the strain has sufficient time to grow,but before it reaches senescence. The yield of DHA of the culture iscalculated. If less than the desired amount, additional subculturing isperformed in the following manner. Fresh low pH media is prepared andinoculated with the low pH cultivated culture, and incubated for anappropriate amount of time. The yield of DHA of the culture iscalculated. If the yield of DHA is less than the desired amount,subculturing is repeated until the desired yield of DHA is achieved. Apreferred pH to select tolerance for is at or below about 6, morepreferably at or below about 5.5, even more preferably at or below about5, and even more preferably at or below 4.5. Media in which to carry outthis method is any culture media known in the art, with the pH adjustedto the desired levels. A preferred media in which to carry out thesubculturing is the media described in Example 1.

The present invention also includes a biomass produced by one of themethods of the invention.

Cultivation conditions consistent with the organisms and methods of thepresent invention may be accomplished by methods known in the art andinclude the methods disclosed in U.S. Pat. No. 5,130,242, U.S. Pat. No.5,407,957, U.S. Pat. No. 5,397,591; U.S. Pat. No. 5,492,938; and U.S.Pat. No. 5,711,983, and optimal conditions may readily be determined bythose skilled in the art. Briefly, cultivation may be accomplished inany suitable fermentor, preferably in either a stirred tank fermentor oran air 111 t fermentor, which provide a source of oxygen to themicroorganisms. The agitation of the microorganism should be maintainedat a level such that while dissolved oxygen concentrations aresufficient to support the growth of the culture and production of DHA,the agitation does not shear or otherwise damage the microorganisms.Preferred levels of dissolved oxygen are at least 10% of air saturationlevel. More preferably, levels of dissolved oxygen are maintained fromabout 10% to about 50% of air saturation levels.

Cultivation may be carried out at any life-sustaining temperature.Generally, microorganisms will grow at temperatures ranging from about15° C. to about 34° C. Preferably the temperature is maintained at about20° C. to about 28° C.

The organisms may be harvested by conventional means, known to those ofskill in the art, such as centrifugation, flocculation, or filtration,and can be processed immediately or dried for future processing. Ineither event, lipid may be extracted. As used herein, the term “lipid”includes phospholipids; free fatty acids; esters of fatty acids;triacylglycerols; diacylglycerides; monoacylglycerides;lysophospholipids; soaps; phosphatides; sterols and sterol esters;carotenoids; xanthophylls (e.g., oxycarotenoids); hydrocarbons; andother lipids known to one of ordinary skill in the art. As is wellunderstood by the skilled artisan, the DHA referred to in the presentinvention can be in the form of these various lipids, and is not limitedto the free fatty acid. Different types or components of the lipids canbe extracted, depending on the extraction technique that is used. Lipidscan be extracted with an effective amount of solvent. Suitable solventscan be determined by those of skill in the art. Polar lipids (e.g.,phospholipids) are generally extracted with polar solvents (e.g.,chloroform/methanol) and neutral lipids (e.g., triacylglycerols) aregenerally extracted with nonpolar solvents (e.g., hexane). A preferredsolvent is pure hexane. A suitable ratio of hexane to dry biomass isabout 4 liters of hexane per kilogram of dry biomass. The hexanepreferably is mixed with the biomass in a stirred reaction vessel at atemperature of about 50° C. for about 2 hours. After mixing, the biomassis filtered and separated from the hexane containing the oil. The hexaneis removed from the oil by distillation techniques known to those ofskill in the art. Conventional oilseed processing equipment is suitableto perform the filtering, separation and distillation. Additionalprocessing steps, known to those of skill in the art, can be performedif required or desirable for a particular application. Alternativemethods for lipid recovery are described in the following referenceswhich are incorporated by reference herein in their entirety: PCTPublication WO 0176715, entitled “Method for the Fractionation of Oiland Polar Lipid-Containing Native Raw Materials”; PCT Publication WO0176385, entitled “Method For The Fractionation Of Oil And PolarLipid-Containing Native Raw Materials Using Alcohol And Centrifugation”;PCT Publication WO 0153512, entitled “Solventless Extraction Process.”

The present invention, while disclosed in terms of specific organismsand methods, is intended to include all such methods and strainsobtainable and useful according to the teachings disclosed herein,including all such substitutions, modifications, and optimizations aswould be available expedients to those of ordinary skill in the art. Thefollowing examples and test results are provided for the purposes ofillustration and are not intended to limit the scope of the invention.

Example 1

This Example describes the preparation of Standard Screening Medium(SSM) with 4.5 g/l NaCl. To prepare the media, the first step includesadding the following compounds to distilled water to 90% of finaldesired volume as shown in Table 1. All compounds are available fromSigma Aldrich, St. Louis, Mo.

TABLE 1 Amounts and Final Concentrations of media before autoclavingAmount Amount Amount chloride potassium sodium Final ion added ion addedion added Compound Concentration (g/l) (g/l) (g/l) CaCl₂—2H₂O¹ 0.3 g/l0.09 MgSO₄—7H₂O 1.25 g/l NaCl 4.5 g/l 3 1.5 MES 10.7 g/l MSG 1.5 g/lTastone 154 0.5 g/l KH₂PO₄ 0.014 g/l .004 KCl 0.14 g/l 0.067 0.073CuSO₄—5H₂O 0.15 × 10⁻³ g/l CoCl₂—6H₂O 0.3 × 10⁻³ g/l negligible H₃BO₃ 10× 10⁻³ g/l MnCl₂—4H₂O 4.5 × 10⁻³ g/l negligible ZnSO₄—7H₂O 0.3 × 10⁻³g/l NaOH (to ad- 1.16 g/l 0.67 just pH to 6.3) FeCl₂ ² 6 × 10⁻³ g/mlnegligible Thiamine³ 1 × 10⁻³ g/l Biotin³ 2 × 10⁻⁶ g/l glucose⁴ 50 g/ltotal of each 3.16 0.08 2.17 ion ¹Calcium chloride dihydrate is 244g/mol with 28.7% chloride. ²stock solution autoclaved separately andadded in a sterile manner to media post-autoclave; made fresh every twoweeks. ³stock solution filter sterilized through a 0.2 micron filter;stored at 4° C. in the dark. Added in a sterile manner to mediapost-autoclave. ⁴stock solution autoclaved separately. Added in asterile manner to media post-autoclave.

Bring autoclaved media up to 100% volume with sterile water. Forscreening experiments, 35 ml of SSM media is added to sterile 250 mlErlenmeyer flasks. 1 ml of inoculum is added per flask for an initialcell concentration of 1×10⁵ cells per mL Inoculum is of 5-6 day oldculture. Cultures are grown at 26.5° C. on a rotary shaker at 135 rpm.

Example 2

This Example describes the preparation of 1000 ppm chloride ionScreening Medium (SSM) with 1.41 g/l NaCl (that together with calciumchloride and potassium chloride results in approximately 1000 ppm, 1 g/lchloride ion). To prepare the media, the first step includes adding thefollowing compounds to deionized distilled water to 90% of final desiredvolume as shown in Table 2. All compounds are available from SigmaAldrich, St. Louis, Mo.

TABLE 2 Amounts and Final Concentrations of media before autoclavingAmount Amount Amount chloride potassium sodium Final ion added ion addedion added Compound Concentration (g/l) (g/l) (g/l) CaCl₂—2H₂O 0.3 g/l0.09 MgSO₄—7H₂O 1.25 g/l NaCl 1.41 g/l 0.85 0.47 MES 10.7 g/l MSG 1.5g/l Tastone 154 0.5 g/l KH₂PO₄ 0.014 g/l 0.004 KCl 0.14 g/l  0.067 0.073CuSO₄—5H₂O 0.15 × 10⁻³ g/l CoCl₂—6H₂O 0.3 × 10⁻³ g/l negligible H₃BO₃ 10× 10⁻³ g/l MnCl₂—4H₂O 4.5 × 10⁻³ g/l negligible ZnSO₄—7H₂O 0.3 × 10⁻³g/l NaOH (to ad- 1.6 g/l 0.67 just pH to 6.3) FeCl₂ ¹ 6 × 10⁻³ g/lnegligible Thiamine² 1 × 10⁻³ g/l Biotin² 2 × 10⁻⁶ g/l glucose³ 50 g/ltotal of each 1.00 0.08 1.14 ion ¹stock solution autoclaved separatelyand added in a sterile manner to media post-autoclave; made fresh everytwo weeks. ²stock solution filter sterilized through a 0.2 micronfilter; stored at 4° C. in the dark. Added in a sterile manner to mediapost-autoclave. ³stock solution autoclaved separately. Added in asterile manner to media post-autoclave.

Bring autoclaved media up to 100% volume with sterile water. Forscreening experiments, 35 ml of SSM media is added to sterile 250 nilErlenmeyer flasks. 1 ml of inoculum is added per flask for an initialcell concentration of 1×10⁵ cells per ml. Inoculum is of 5-6 day oldculture. Cultures are grown at 26.5° C. on a rotary shaker at 135 rpm.

Example 3

This Example describes the preparation of 300 ppm chloride ion ScreeningMedium (SSM) with 0.211 g/l NaCl (that together with calcium chlorideand potassium chloride results in 0.3 g/l chloride ion). To prepare themedia, the first step includes adding the following compounds todeionized distilled water to 90% of final desired volume as shown inTable 3. All compounds are available from Sigma Aldrich, St. Louis, Mo.

TABLE 3 Amounts and Final Concentrations of media before autoclavingAmount Amount Amount chloride potassium sodium Final ion added ion addedion added Compound Concentration (g/l) (g/l) (g/l) CaCl₂—2H₂O 0.3 g/l0.09 MgSO₄—7H₂O 1.25 g/l NaCl 0.211 g/l 0.13 0.07 MES 10.7 g/l MSG 1.5g/l Tastone 154 0.5 g/l KH₂PO₄ 0.014 g/l 0.004 KCl 0.14 g/l  0.067 0.073CuSO₄—5H₂O 0.15 × 10⁻³ g/l CoCl₂—6H₂O 0.3 × 10⁻³ g/l negligible H₃BO₃ 10× 10⁻³ g/l MnCl₂—4H₂O 4.5 × 10⁻³ g/l negligible ZnSO₄—7H₂O 0.3 × 10⁻³g/l NaOH (to ad- 1.16 g/l 0.67 just pH to 6.3) FeCl₂ ¹ 6 × 10⁻³ g/lnegligible Thiamine² 1 × 10⁻³ g/l Biotin² 2 × 10⁻⁶ g/l glucose³ 50 g/ltotal of each 0.30 0.08 0.74 ion ¹stock solution autoclaved separatelyand added in a sterile manner to media post-autoclave; made fresh everytwo weeks. ²stock solution filter sterilized through a 0.2 micronfilter; stored at 4° C. in the dark. Added in a sterile manner to mediapost-autoclave. ³stock solution autoclaved separately. Added in asterile manner to media post-autoclave.

Bring autoclaved media up to 100% volume with sterile water. Forscreening experiments, 35 ml of SSM media is added to sterile 250 mlErlenmeyer flasks. 1 ml of inoculum is added per flask for an initialcell concentration of 1×10⁵ cells per ml. Inoculum is of 5-6 day oldculture. Cultures are grown at 26.5° C. on a rotary shaker at 135 rpm.

Example 4

This Example describes the procedure for Growth and Harvest ofCrypthecodinium cohnii in pH 6.3 SSM.

SSM media was prepared as described in one of Examples 1-3, depending onwhich media was being tested. Additional media components were preparedand added to a media as described in Examples 1-3. All steps prior toharvest were carried out in sterile conditions.

To prepare the inoculum culture, the following procedures were used. Toa 250 ml Erlenmeyer flask, 49 ml of SSM (described in Example 1) wereadded to a 250 ml Erlenmeyer flask. 1 ml of a five day old culture of C.cohnii Strain T-HF (Strain T-HF identifies the organism ATCC 40750 thathas been repeatedly cultured) was added. The culture flask was placed ona shaker rotating at 135 rpm in a 27° C. incubator with no lights. Afterthree days of growth, the culture is moved to a sterile hood and 1 ml isremoved and counted using a Coulter Counter (Coulter Z2 Particle Countand Size Analyzer, obtained from Beckman Coulter, Inc.). The cell countis used to calculate the amount of the inoculum culture that must beused to start a new 50 ml culture at a cell density of 1.0×10⁵ cells perml.

To test the different media components, an appropriate media wasprepared as described below and introduced into a sterile 250 mlErlenmeyer flask. The amount of inoculum as previously calculated wastransferred into the culture flask containing the media prepared in theErlenmeyer flask. The culture flask was placed on a shaker rotating at135 rpm in a 27° C. incubator with no lights. After seven days ofgrowth, the culture was harvested as follows.

A 50 ml centrifuge tube (obtained from VWR Scientific) was labeled andweighed for each culture. Another 50 ml centrifuge tube was labeled, butnot weighed, for each culture. The culture was then poured into thelabeled 50 ml tube. Volumes were recorded and cell counts performed withthe Coulter Z2 Particle Count and Size Analyzer. The pH was measured.

Half the culture was poured into the tared 50 ml tube, and a 70%solution of isopropyl rubbing alcohol (IPA) was added to bring the totalvolume in the tube to 50 ml. The culture was mixed by inverting the tubetwo to three times. The culture was then centrifuged at 4000 rpm forfive minutes using a Sorvall General Purpose RC-3 Centrifuge. Thesupernatant was poured off. The other half of the culture was poured ontop of the pellet and the steps were repeated starting with the 70%solution of the TPA. The pellet was then washed two times with 39% IPAusing the following procedure: to cell pellet, add 35 mL 39% IPA; vortexthe tube (using a Vortex Genie-2 from VWR Scientific) at full speed for10 seconds; after collection, the pellet was then freeze-dried for atleast 48 hours.

The tube containing the pellet (biomass) was weighed and the dry weightof the biomass was calculated. Dry weight was calculated as follows:determine the weight of the tube containing the biomass minus the tareweight of the tube. Divide this number by the recorded volume of cultureat harvest, divided by 1000.

Fatty acid composition (and % DHA) may be determined according toprocedures disclosed in Morrison and Smith, “Preparation of Fatty AcidMethyl Esters and Dimethylacetals from Lipids with BoronFluoride-Methanol”, Journal of Lipid Research, Vo. 5, 1964, and theAmerican Oil Chemist's Society Official Methods used to quantitate longchain fatty acids and eicosapentaenoic acid (EPA) and DHA in marine oils(Method CeIb-89). Briefly, the samples are mixed with standard amountsof oil (internal standards), saponified with 0.5 N methanolic sodiumhydroxide, and derivatized with boron trifluoride/methanol. The fattyacid methyl esters are extracted and analyzed on a gas chromatographwith frame ionization detector (Hewlett Packard 5890 Series II Plus gaschromatograph using a 30 m×0.25 mm×0.25 μm Restek FAMEWAX #12497column).

Example 5

This Example describes the growth of C. cohnii and the production of DHAat low NaCl levels using prior art media.

One liter of SSM not containing NaCl was made and autoclaved. Fourstocks of concentrated NaCl were prepared (135 g/l, 90 g/l, 45 g/l, and22.5 g/l). To each shake flask containing 48.75 ml of SSM minus NaClmedia and 1.25 ml of the appropriate NaCl stock was added. Two controlswere set up: 4.5 g/l NaCl using normal SSM as described in Example 1,and no NaCl using SSM with no added NaCl. Duplicates of each NaCl levelwere used.

Growth and harvest was performed as described in Example 4. Table 5describes the results of this Example. All numbers are given as anaverage of two cultures.

TABLE 5 Biomass, % DHA, % Fat, and DHA yield for C. cohnii grown in SSMcontaining lowered amounts of NaCl. g/l g/l chloride biomass dry % DHA %fat in NaCl ion¹ weight g/l in fat (wt/wt) biomass (wt/wt) 4.5 2.73 3.5351.63 52.45 3.38 2.05 3.66 51.55 47.83 2.25 1.37 3.85 52.19 48.40 1.730.68 2.73 54.65 54.59 0.56 0.34 2.70 55.48 48.81 0 0 1.99 51.00 34.19¹Reflects amount of chloride ion (0.20 g/l) from sodium chloride only.See Examples 1-3.

Table 5 shows the yield of biomass, % fat, and DHA yield for C. cohniigrown in SSM containing lowered amounts of NaCl. It can be seen that asthe amount of NaCl added into the culture decreases, both biomass yieldand fat levels decreased, resulting in a lowered yield of DHA.

Example 6

This Example describes the yield of DHA achieved with 4.5 g/l NaCl inthe culture medium described in Example 1.

Cultures were grown as described in Example it Table 6 shows the resultsof this Example.

TABLE 6 Biomass, % DHA, % Fat, and DHA yield for C. cohnii grown in SSMof Example 1 (g/l) (g/l) (g/l) (g/l) % DHA % fat in biomass sodiumsodium chloride¹ sodium in fat biomass dry weight chloride sulfate ionion (wt/wt) (wt/wt) (g/l) 4.5 g/l 2.73 1.77 53.9 65.03 3.1 ¹Reflectsamount of chloride ion from sodium chloride only

Example 7

This Example describes enhanced growth of C. cohnii and production ofDHA in low chloride media using various concentrations of potassium ionand sodium ion in the form of potassium sulfate and sodium sulfate.

Low chloride SSM was prepared in the manner described in Example 3,using 0.18 g/l calcium acetate and omitting calcium chloride andpotassium chloride. Every possible combination of K₂SO₄ concentrationsof 0.16 g/l, 0.80 g/l, 1.6 g/l, 3.2 g/l, and 4.8 g/l were tested againstNa₂SO₄ concentrations of 4.9 g/l, 9.8 g/l, 14.7 g/l, 19.6 g/l, and 24.5g/l using a two dimensional matrix. All cultures were grown as describedin Example 4. The results are presented in Table 7.

TABLE 7 Comparison of the biomass, % DHA, % Fat, and DHA yield obtainedfor C. cohnii grown in media with varying concentrations of potassiumsulfate and sodium sulfate % Sodium % DHA Fat in g/L g/L ion¹ PotassiumDW in fat biomass Flask K₂SO₄ Na₂SO₄ (g/l) ion (g/l) g/L (wt/wt) (wt/wt)1 0.16 4.90 1.77 0.07 2.55 57.97 60.80 2 0.16 9.80 3.35 0.07 1.53 52.3941.45 3 0.16 14.70 4.93 0.07 — — — 4 0.16 19.60 6.53 0.07 0.75 42.8813.28 5 0.16 24.50 8.11 0.07 0.71 41.46 12.11 6 0.80 4.90 1.77 0.36 3.7956.76 63.19 7 0.80 9.80 3.35 0.36 4.03 55.11 64.96 8 0.80 14.70 4.930.36 3.66 55.14 64.39 9 0.80 19.60 6.52 0.36 3.07 56.88 58.12 10 0.8024.50 8.11 0.36 2.91 57.37 53.65 11 1.60 4.90 1.77 0.72 3.74 55.90 63.4612 1.60 9.80 3.35 0.72 3.83 55.00 65.43 13 1.60 14.70 4.93 0.72 3.4956.48 60.09 14 1.60 19.60 6.53 0.72 3.18 54.71 54.92 15 1.60 24.50 8.110.72 2.83 54.82 49.02 16 3.20 4.90 1.77 1.44 3.51 54.42 63.99 17 3.209.80 3.35 1.44 3.36 55.40 61.12 18 3.20 14.70 4.93 1.44 3.40 55.61 59.3419 3.20 19.60 6.53 1.44 3.07 57.07 59.44 20 3.20 24.50 8.11 1.44 2.7757.00 57.07 21 4.80 4.90 1.77 2.15 2.82 54.94 57.43 22 4.80 9.80 3.352.15 2.81 53.97 58.12 23 4.80 14.70 4.93 2.15 2.94 54.26 58.75 24 4.8019.60 6.52 2.15 2.82 55.53 56.88 25 4.80 24.50 8.11 2.15 2.50 57.0253.00 ¹Includes sodium ion added by 0.45 g/l sodium chloride or 0.18 g/lsodium ion.

The results shown in Table 7 indicate that increased potassium levelscaused the growth and yield of DHA for C. cohnii to be comparable tothat achieved at high chloride levels. The enhancement effect in thisExample appeared at 0.8 g/l potassium sulfate, the second-lowest leveltested, and thereafter was relatively insensitive to the amounts ofpotassium sulfate. At the highest levels of potassium sulfate tested,4.8 g/l, there appeared to be a slight decline in yield. Growth and DHAyield also appeared relatively insensitive to the amount of sodiumsulfate used, however, growth and yield dropped slightly as increasingamounts of sodium sulfate were used, starting at about 19.6 g/l sodiumsulfate. The best combinations based on the amount of DHA in g/L werethose using: 0.8 g/L K₂SO₄ and 9.8 g/L Na₂SO₄, representing a 5×increase of potassium and a 2× increase of sodium over the normal LowChloride-SSM (described in Example 3); and 1.6 g/L K₂SO₄ and 9.8 g/LNa₂SO₄, representing a 10× increase of potassium and a 2× increase ofsodium over the normal Low chloride-SSM (described in Example 3).

Example 8

This Example demonstrates enhancement of growth of C. cohnii andproduction of DHA using media containing a range of potassium sulfate,0.32 g/l, 0.64 g/l, 0.96 g/l, 1.28 g/l, 1.60 g/l, and 1.9 g/l and sodiumsulfate at 4.9 g/l and 9.8 g/l.

Low chloride SSM was prepared in the manner described in Example 7, andall cultures were grown as described in Example 4. The results arepresented in Table 8.

TABLE 8 Comparison of the biomass, % DHA, % Fat, and DHA yield obtainedfor C. cohnii grown in media with varying concentrations of potassiumsulfate and sodium sulfate % % DHA Fat in Sodium g/L g/L DW in fatbiomass ion¹ Potassium Flask K₂SO₄ Na₂SO₄ g/L (wt/wt) (wt/wt) (g/l) ion¹(g/l) 1 0.32 4.90 3.22 57.76 75.22 1.77 0.14 2 0.32 9.80 3.05 57.6166.15 3.35 0.14 3 0.64 4.90 3.49 58.66 61.45 1.77 0.29 4 0.64 9.80 3.4758.50 63.22 3.35 0.29 5 0.96 4.90 3.43 58.45 59.98 1.77 0.43 6 0.96 9.803.66 51.91 58.03 3.35 0.43 7 1.28 4.90 3.51 58.72 58.67 1.77 0.57 8 1.289.80 3.67 56.93 75.09 3.35 0.57 9 1.60 4.90 3.32 57.16 65.76 1.77 0.7210 1.60 9.80 3.57 56.89 62.11 3.35 0.72 11 1.90 4.90 3.36 56.15 59.951.77 0.85 12 1.90 9.80 3.54 54.74 60.42 3.35 0.085 ¹Includes sodium ionadded by 0.45 g/l sodium chloride or 0.18 g/l sodium ion.

Results shown in Table 8 Showed that the optimum DHA yield occurs withconcentrations of K₂SO₄ at 1.28 g/L, and 9.8 g/L Na₂SO₄. The resultsshown in Table 8 indicate that the effect of additional potassium can beseen at potassium sulfate levels as low as 0.32 g/l and appearrelatively constant through 1.90 g/l. Growth and yield are relativelyinsensitive to sodium sulfate levels of 4.9 g/l or 9.8 g/l.

Example 9

The following Example describes subculturing C. cohnii to obtain astrain that is adapted to growth at pH 5.

C. cohnii Strain T-HF was cultured in shake flasks in the mannerdescribed in Example 4, in media described in Example 1, except that thepH of the media upon the start of culture was pH 5. After 7 days, aninoculum from the culture was used to start a new culture at pH 5 underthe same conditions. Initially the growth at pH 5 was slow, but aftermultiple transfers, the DHA yield began to improve and over time hasapproached the yield seen from cultures grown at pH 6.3, resulting in alow pH strain. See FIG. 1. It was noted that the pH of the culture atthe end of the 7 day growth period was 5.4. Attempts to adapt the strainusing buffers citrate, malate, acetate, and lactate were unsuccessfuldue to those buffer's toxic effect on Strain THF.

The low pH strain was then grown in the pH 5 media described above, butpH was kept at 5.0. The low pH adapted strain grew equally well at pH 5and at pH 5.4.

Example 10

The following Example describes a comparison of DHA yields from C.cohnii Strain T-HF grown at pH 6.3 and 2730 ppm chloride ion media andthe low pH strain at pH 5 in 1000 ppm chloride ion media.

C. cohnii Strain T-HF was grown as described in Example 4 in media asdescribed in Example 1. C. cohnii strain was grown in low chloride mediaas described in Example 2, with potassium sulfate and sodium sulfateadded. Each experiment was run in duplicate, and all flasks wereharvested daily to determine the kinetics of DHA yield. The results areshown in FIG. 1. FIG. 1 shows that the kinetics of DHA yield were nearlyidentical under the two different media conditions.

Example 11

The following Example describes efforts to adapt C. cohnii Strain T-HFto growth at pH 4.5 in 2730 ppm chloride ion medium.

C. cohnii Strain T-HF was grown in the manner described in Example 10,with the exception that the media was adjusted to pH 4.5 and half of theMSG was replaced with lysine, while maintaining a constant level oforganic nitrogen in the media.

After repeated culturing, yields of DHA of about one-third of yieldsseen at pH 5 or pH 6.3 were obtained.

Example 12

The following Example describes efforts to define conditions for C.cohnii growth and DHA yields at pH 4.5 by manipulating potassiumconcentrations.

A. Factorial experiments were performed at pH 4.5 at 2.73 g/l chlorideion to assess the effect of potassium ion (0.16 g/l to 3.2 g/l). Theresults showed that higher levels of potassium ion increased the DHAyield to approximately two-thirds of yields obtained for C. cohnii at pH6.3 with the media as described in Example 1.

B. A factorial experiment was run in the manner described in Part A,above, with the exception that the chloride ion levels were heldconstant at 1.0 g/l. The results showed that higher levels of potassiumion increased the DHA yield to approximately two-thirds of yieldsobtained for C. cohnii grown at pH 6.3 with the media as described inExample 1. The DHA yields obtained at 2.73 g/l chloride ion (describedin Part A, above) and at 1.0 g/l chloride ion were comparable.

Example 13

This Example describes a time course experiment comparing yields of DHAobtained using the pH 4.5 strain described in Example 12 and Strain T-HFat pH 6.3, 1.0 g/l chloride ion.

The pH 4.5 strain of Example 12 was grown in low chloride, pH 4.5 mediaas specified in Table 9 in shake flasks after the manner described inExample 4. C. cohnii Strain T-HF was grown in the manner described inExample 4 using the media described in Example 1. Inoculum for the pH4.5 experiment was prepared at pH 4.5, and amounts of inoculum wereestimated due to clumping of the cells at pH 4.5.

TABLE 9 Low chloride, pH 4.5 media Amount Amount Amount chloridepotassium sodium Final ion added ion added ion added CompoundConcentration (g/l) (g/l) (g/l) CaCl₂—2H₂O 0.3 g/l 0.09 MgSO₄—7H₂O 1.25g/l NaCl 1.41 0.86 0.55 MES 10.7 g/l MSG 0.75 g/l Tastone 154 0.5 g/lLysine-HCl 0.37 KH₂PO₄ 0.014 g/l 0.004 K₂SO₄ 0.15 0.07 Na₂SO₄ 3.46 1.12CuSO₄—5H₂O 0.15 × 10⁻³ g/l H₃BO₃ 10 × 10⁻³ g/l MnCl₂—4H₂O 4.5 × 10⁻³ g/lnegligible ZnSO₄—7H₂O 0.3 × 10⁻³ g/l NaOH (to ad- 1.16 g/l 0.67 just pHto 6.3) FeCl₂ ¹ 6 × 10⁻³ g/l negligible Thiamine² 1 × 10⁻³ g/l Biotin² 2× 10⁻⁶ g/l glucose³ 50 g/l total of each 0.30 0.08 0.74 ion

Flasks were harvested daily to determine the kinetics of the DHA yield.The results of the experiment (FIG. 2) indicated that the DHA yield atpH 4.5 was always lower than at pH 6.3, but the rate of increase in theDHA yield as a function of time was about the same at each pH. Thissuggests that at pH 4.5 the culture is able to accumulate DHA at thesame rate as the culture at pH 6.3, however, a lag existed in the DHAyield in cultures grown at pH 4.5 compared to pH 6.3.

This result shows that given extra time, i.e. approximately 24 hours,the DHA yield at pH 4.5 was the same as at pH 6.3. It was not clearwhether the lag was caused by a delay in the DHA accumulation at pH 4.5,and as a result, the pH 4.5 culture always had a DHA yield that waslower than the pH 6.3 culture of the same age, or whether the lag wascaused by the pH 4.5 culture not receiving an equivalent amount ofinoculum. At pH 4.5, the Strain T-HF cells clump such that it is notpossible to get an accurate cell count of the culture, and the amount ofinoculum to use must be estimated. Therefore, it is possible that the pH4.5 culture received less inoculum and therefore caused an apparent lagin the kinetics of DHA yield.

Regardless, these data indicated that by using the low pH adapted C.cohnii strain and the instant culture media at pH 4.5, the same DHAyield can be achieved as the culture medium at pH 6.3, if culturing timeis extended.

Example 14

Further optimization of the ion concentrations described in Example 13above and further subculturing of C. cohnii Strain T-HF which has beenadapted to pH 5, using techniques described in Example 10, are carriedout to decrease the lag time, resulting in 7-day DHA yields at pH 4.5which are comparable to the yields obtained for C. cohnii grown at pH6.3 with the media as described in Example 1.

The principles, preferred embodiments and modes of operation of thepresent invention have been described in the foregoing specification.The invention which is intended to be protected herein should not,however, be construed as limited to the particular forms disclosed, asthese are to be regarded as illustrative rather than restrictive.Variations and changes may be made by those skilled in the art withoutdeparting from the spirit of the present invention. Accordingly, theforegoing best mode of carrying out the invention should be consideredexemplary in nature and not as limiting to the scope and spirit of theinvention as set forth in the appended claims.

1.-21. (canceled)
 22. A method of producing docosahexaenoic acid (DHA),comprising: culturing a heterotrophic microalga in a culture medium,wherein the culture medium comprises: chloride ion at a concentration ofless than or equal to 3 g/potassium potassium ion at a concentration ofgreater than or equal to 0.5 g/L; sodium ion at a concentration of 1 g/Lto 8 g/L; wherein the pH is less than or equal to 5; and wherein a ratioof sodium ion to potassium ion is less than or equal to 27:1; andrecovering DHA-containing lipid from the microalga, wherein themicroalga produces at least 0.04 g DHA per 10⁹ cells.
 23. The method ofclaim 22, wherein the concentration of chloride ion is less than orequal to 2 g/L.
 24. The method of claim 22, wherein the concentration ofchloride ion is less than or equal to 1 g/L.
 25. The method of claim 22,wherein the concentration of chloride ion is less than or equal to 0.3g/L.
 26. The method of claim 22, wherein the concentration of sodium ionis 1.5 g/L to 5 g/L.
 27. The method of claim 22, wherein the pH is 4.5.28. The method of claim 22, wherein a source of potassium comprisespotassium sulfate.
 29. The method of claim 22, wherein the microalgaproduces at least 0.10 g DHA per 10⁹ cells.
 30. The method of claim 22,wherein the microalga produces at least 0.20 g DHA per 10⁹ cells. 31.The method of claim 22, wherein a ratio of sodium ion to potassium ionis less than or equal to 15:1.
 32. The method of claim 22, wherein aratio of sodium ion to potassium ion is less than or equal to 4:1. 33.The method of claim 22, further comprising recovering DHA from theDHA-containing lipid.
 34. A biomass produced by the method of claim 22.35. A lipid extracted from the biomass of claim
 34. 36. The method ofclaim 22, wherein the microalga is of the class Dinophyceae.
 37. Themethod of claim 22, wherein the microalga is of the genusCrypthecodinium.
 38. The method of claim 22, wherein the microalga is ofthe species Crypthecodinium cohnii.